Dian fluorescence intensity (MFI) was obtained from single parameter histograms. Comparisons between groups was accomplished by single parameter t-test for differences between means and the Wilcoxon Signed-Rank test for differences in medians. A result was considered significant at a p value of 0.05.Results NKG2D Ligands are transiently upregulated on TMZresistant U87MG glioma cells after exposure to TMZIn this experiment, we sought to determine if TMZ exposure stresses a TMZ-resistant U87 culture that had not previously been exposed to TMZ, NKG2D ligand expression was examined at incremental time intervals following TMZ exposure. ChemotherDrug Resistant cd T Cell ImmunotherapyFigure 1. Transitory increase in stress-associated antigens on TMZ-resistant cell line U87 after exposure to TMZ. U87 cells were cultured to confluence and incubated in media containing 100 mM TMZ. Stress antigens were order 223488-57-1 assessed at the time intervals noted on the x axis by flow cytometry and the increase in median fluorescence intensity over isotype control were calculated. Data are shown as percentage increase over unmanipulated U87 cells. SD of 3 experiments are shown. doi:10.1371/journal.pone.0051805.gGene-modified cd T cells function in the presence of 
TMZCytolytic function of MGMT-modified cd T cells was evaluated against TMZ-resistant clones of SNB-19 and U373 in the presence of TMZ using the MedChemExpress Dimethylenastron Cytotoxicity assay procedure described above but modified to include 100 mM TMZ during the four-hour incubation. TMZ-resistant clones from both cell lines propagated slowly in TMZ-supplemented media but were highly resistant to the drug. As proof-of-concept, cytotoxicity against SNB-19TMZ-R cells was assessed in separate experiments from U373TMZ-R cells 15481974 using different donors in order to conserve available cells while conducting the experiment in such a manner as to determine if expanded/activated cdTMZ-R function was consistent across donors and cell lines. TMZ-resistant clones remained viable in the absence of TMZ for the length of the assay as shown in the upper panels of Figure 5a and 5b. When both cell lines were incubated in the presence of TMZ and expanded/activated cdTMZ-R, viability as measured by the uptake of the dye ToPro Iodide was noticeably increased after four hours in culture, as shown in the lower panels of Figure 5a and 5b. Dose-dependent cytotoxicity of WT cd was significantly less when assayed against SNB-19TMZ-R (c) with no TMZ in the media vs. cdTMZ-R against SNB-19TMZ-R in the presence of TMZ (p = 0.0085). Cytotoxicity was also trended greater against TMZ-resistant U373 with cdTMZ-R as effectors as well when the assay was conducted in the presence of TMZ (p = .0875) but did not achieve significance at the p = 0.05 level. These assays were conducted as separate experiments from different donors.DiscussionNo treatment options are currently available to 
control the progression of rapidly proliferating invasive high-grade gliomas. Intensive chemotherapeutic strategies, such as high dose temozolomide can lead to lymphodepletion, impaired T cell function, and consequent suppression of anti-tumor immune responses [34]. We have previously shown that local placement of allogeneic cd T cells can slow progression of small established intracranial tumors and significantly extend survival in a human GBM xenograft model [35]. The characteristically rapid growth of high-grade gliomas as well as both systemic and local immunosuppression, however, remain formidabl.Dian fluorescence intensity (MFI) was obtained from single parameter histograms. Comparisons between groups was accomplished by single parameter t-test for differences between means and the Wilcoxon Signed-Rank test for differences in medians. A result was considered significant at a p value of 0.05.Results NKG2D Ligands are transiently upregulated on TMZresistant U87MG glioma cells after exposure to TMZIn this experiment, we sought to determine if TMZ exposure stresses a TMZ-resistant U87 culture that had not previously been exposed to TMZ, NKG2D ligand expression was examined at incremental time intervals following TMZ exposure. ChemotherDrug Resistant cd T Cell ImmunotherapyFigure 1. Transitory increase in stress-associated antigens on TMZ-resistant cell line U87 after exposure to TMZ. U87 cells were cultured to confluence and incubated in media containing 100 mM TMZ. Stress antigens were assessed at the time intervals noted on the x axis by flow cytometry and the increase in median fluorescence intensity over isotype control were calculated. Data are shown as percentage increase over unmanipulated U87 cells. SD of 3 experiments are shown. doi:10.1371/journal.pone.0051805.gGene-modified cd T cells function in the presence of TMZCytolytic function of MGMT-modified cd T cells was evaluated against TMZ-resistant clones of SNB-19 and U373 in the presence of TMZ using the cytotoxicity assay procedure described above but modified to include 100 mM TMZ during the four-hour incubation. TMZ-resistant clones from both cell lines propagated slowly in TMZ-supplemented media but were highly resistant to the drug. As proof-of-concept, cytotoxicity against SNB-19TMZ-R cells was assessed in separate experiments from U373TMZ-R cells 15481974 using different donors in order to conserve available cells while conducting the experiment in such a manner as to determine if expanded/activated cdTMZ-R function was consistent across donors and cell lines. TMZ-resistant clones remained viable in the absence of TMZ for the length of the assay as shown in the upper panels of Figure 5a and 5b. When both cell lines were incubated in the presence of TMZ and expanded/activated cdTMZ-R, viability as measured by the uptake of the dye ToPro Iodide was noticeably increased after four hours in culture, as shown in the lower panels of Figure 5a and 5b. Dose-dependent cytotoxicity of WT cd was significantly less when assayed against SNB-19TMZ-R (c) with no TMZ in the media vs. cdTMZ-R against SNB-19TMZ-R in the presence of TMZ (p = 0.0085). Cytotoxicity was also trended greater against TMZ-resistant U373 with cdTMZ-R as effectors as well when the assay was conducted in the presence of TMZ (p = .0875) but did not achieve significance at the p = 0.05 level. These assays were conducted as separate experiments from different donors.DiscussionNo treatment options are currently available to control the progression of rapidly proliferating invasive high-grade gliomas. Intensive chemotherapeutic strategies, such as high dose temozolomide can lead to lymphodepletion, impaired T cell function, and consequent suppression of anti-tumor immune responses [34]. We have previously shown that local placement of allogeneic cd T cells can slow progression of small established intracranial tumors and significantly extend survival in a human GBM xenograft model [35]. The characteristically rapid growth of high-grade gliomas as well as both systemic and local immunosuppression, however, remain formidabl.